The long-term goal of this grant is to fully understand the biosynthesis, function, and clinical importance of a key molecule in the endoplasmic reticulum (ER) of mammalian cells, the lipid-linked oligosaccharide (LLO) glucose3mannose9N-Acetylglucosamine2-P-P-dolichol, or G3M9Gn2-P-P-Dol. This work is important: i- G3M9Gn2-P-P-Dol synthesis involves novel and exciting biochemical processes yielding insights into other pathways, ii- Once transferred to protein, the G3M9Gn2 unit is processed by a series of enzymes (glycosidases). The various oligosaccharide products play roles in protein folding and degradation ("quality control") in the ER. iii- Genetic defects in the synthesis of G3M9Gn2-P-P-Dol form a family of at least seven human genetic disorders called Congenital Disorders of Glycosylation (CDG) Type la-lg. This application will take advantage of two recent technical innovations from the current funding period: fluomphore-assisted carbohydrate electrophoresis (FACE) to study LLOs, and streptolysin-O (SLO)-based in vitro systems that faithfully preserve in vivo regulatory processes. These two techniques have dramatically changed the way we study and think about this pathway. The application will also bring together new insights into the roles of the Unfolded Protein Response (UPR), the Lec35 protein, and two functionally distinct pools of dolichoI-P (DoI-P) in control of LLO synthesis. These are especially important for the regulation of the initiating step of LLO synthesis, catalyzed by GIcNAc-1-P transferase (GPT). AIM 1: Use the SLO in vitro system to determine the route for recycling of the primary pool of DoI-P and the membrane topology the secondary pool of DoI-P. AIM 2: Test the hypothesis that triggering of the Unfolded Protein Response by robust ER stress leads to a dual mechanism for inhibition of LLO synthesis involving both PERK and Lec35p. AIM 3: Test the hypotheses that GPT interacts directly with Lec35p, and that overexpression of GPT causes defective LLO synthesis by interfering with Lec35p function in a dominant-negative manner. AIM 4: Test the hypothesis that novel sugar-P-dolichols and novel sugar-P-dolichol dependent glycoconjugates remain to be identified in animal cells. Use the unique properties of Lec35 cells and the advantages of the SLO system to identify these glycoconjugates.